Genotoxicant accumulation and cellular defence activation in bivalves chronically exposed to waterborne contaminants from the Seine River

The aim of the present work was to investigate genotoxicant accumulation and biological responses of zebra mussels and blue mussels collected along a pollution gradient in the Seine estuary and in the Seine Bay. The sampling area included three contaminated and one reference sites for each species. The study focused on polyaromatic hydrocarbons (PAH), lindane, polychlorobiphenyls (PCB) and metals known to be potential genotoxicants and/or reactive oxygen species (ROS) inducers. Enzymatic activities related to cellular defence systems including the phase II enzyme glutathione S-transferase (GST) and three antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured in gills. DNA adducts and DNA strand breaks (Comet assay) were measured in digestive gland and hemocytes, respectively. Species differences were observed in metal accumulation (As and Pb), GPx activity and DNA adduct formation. A marked upstream-downstream gradient was reported for PAH body burden and to a lesser extent for PCB and metals with the highest values measured just downstream the industrialized area of Rouen. GST and SOD activities in gills of bivalves were positively related to PAH and metals body burden, respectively. Activation of those cellular defences may prevent accumulation of electrophilic metabolites and free radicals and thus may protect DNA and others macromolecules against oxidation and adduction. Although DNA strand breaks and bulky adducts were detected in both species, levels were relatively low and no significant site differences were observed in June 2003. Our results indicate a clear relationship between genotoxicant accumulation and positive activation of detoxification and antioxidant systems but poor consequences in term of DNA damage for wild population of mussels inhabiting the Seine estuary.     

Tissue differences,dose-response relationship and persistence of DNA adducts in blue mussels (Mytilus edulis L) exposed to benzo[a]pyrene

Baltic Sea blue mussels (Mytilus edulis) were experimentally exposed to the genotoxic model substance benzo[a]pyrene (B[a]P) to study DNA adduct formation. The specific aims were (a). to examine where in the mussels the DNA adducts were formed, in gills or digestive gland; (b). to study the dose-response relationship between B[a]P exposure and DNA adduct formation; and (c). to examine the persistence of the formed adducts. A Scope for growth (SFG) study was also run to compare physiological responses of the mussels with the degree of DNA adduct formation. In an initial dose-response experiment, the mussels were exposed to 0, 5, 50, and 100 microg/l of tritium labelled B[a]P under semi-static conditions for 4 days, and thereafter the bioaccumulation of B[a]P and DNA adduct formation in different tissues was determined using liquid scintillation counting and 32P-postlabelling analysis, respectively. In a following exposure-depuration experiment, mussels were exposed to 17 microg/l of radiolabelled B[a]P under semi-static conditions for 6 days. B[a]P accumulation and DNA adduct formation were determined during the exposure, and B[a]P elimination and persistence of DNA adducts were studied during 28 days of depuration in uncontaminated water. The results revealed large tissue differences in DNA adduct formation. DNA adduct levels were not elevated in the digestive gland of the mussels at any exposure concentration (0-100 microg/l), even though the highest B[a]P tissue concentrations were found in the digestive gland (1.0+/-0.1 mg B[a]P/g tissue dry wt at 100 microg/l, mean+/-SE, n=12). DNA adducts were on the other hand formed in the gills, with the highest levels found in mussels exposed to 50 and 100 microg B[a]P/l, and a dose dependent increase in adduct levels (from 1.6 to 5.9 nmol adducts/mol nucleotides) from 0 to 50 microg B[a]P/l. In gills, DNA adduct levels increased with time during the 6-day exposure period in the exposure-depuration experiment, and then persisted for at least 2 weeks after exposure cessation while B[a]P tissue levels exhibited a rapid decrease (half-life of 8 days). No significant differences were observed in SFG between the control and exposed groups. Since DNA adducts exhibited a relatively high persistence in gills compared to B[a]P tissue concentrations, they seem to be a more integrated measure of genotoxic exposure than only chemical analysis of the contaminant bioaccumulation. The results also suggest that if using analysis of DNA adducts in M. edulis for monitoring purposes, analysis of gills in addition to the more commonly used digestive gland should be taken into consideration.     

Enzymatic biomarker measurement and study of DNA adduct formation in benzo

The aim of this study was to improve the knowledge on the metabolic pathways involved in benzo[a]pyrene (B[a]P) activation and on the relationship between adduct levels and enzymatic biomarker activities. With this purpose, a model to assess pollutant exposure via food supply has been developed for the sentinel organism, Mytilus galloprovincialis. Mussels were fed for 4 weeks with B[a]P-contaminated feed (50 mg/kg dry weight mussel). Bioaccumulation was studied by determination of B[a]P concentration in whole mussel by GC/MS analysis. Different biomarkers of pollutant exposure were measured to assess the metabolic state of the exposed organisms. CYP1A-like immunopositive protein titration and B[a]P hydroxylase (BPH) activity were assessed as indicators of phase I biotransformation. Glutathione-S-transferase (GST) activity was measured as an indicator of the conjugation activities. Catalase (CAT) and DT-diaphorase (DTD) activities were assessed as potential biomarkers of oxidative stress, whereas acetylthiocholine esterase (AChE) activity was measured as an indication of possible neurotoxicity of B[a]P exposure. DNA adduct levels were determined in digestive gland DNA by applying the 32P-postlabeling technique with nuclease P1 enhancement. For the developed conditions of exposure, B[a]P concentration reached in whole mussel tissues was very high (>500 mg/kg d.w. mussel) and significant B[a]P-induced changes were recorded for each enzymatic biomarkers. BPH and CAT activities were significantly increased by B[a]P exposure, whereas GST in the gills, DTD and AChE were significantly depressed. On the other hand, no change in CYP1A-like immunopositive protein content was observed. Induction and increase with time of bulky B[a]P-related DNA adducts were demonstrated in the digestive gland, although at low levels (0.269+/-0.082 adduct/10e8 dNps at maximum) by the 32P-postlabeling assay. DNA adduct level was significantly correlated with whole mussel tissue B[a]P concentration, so were all the enzymatic biomarkers measured except to GST activity in both gill and digestive gland tissues. BPH, DTD, CAT and AChE displayed a strong correlation with adduct levels. These results demonstrate the neurotoxicity and the genotoxicity of B[a]P exposure in the mussel. The induction of bulky DNA adducts in mussels demonstrates the existence of activation pathways already identified in vertebrates. It validates also the suitability of this model for further studies on B[a]P metabolism in mussels. Our results support the proposal of BPH, AChE, DTD and CAT activities as suitable biomarkers of PAH exposure for these sentinel species.     

An in vivo study of benzene metabolite DNA adduct formation in liver of male New Zealand rabbits

Rabbits were treated with benzene (586 mg/kg/ b.i.d./4 days) after which DNA was isolated from liver and analyzed for adduct formation using the [³²P] postlabeling method of Randerath and coworkers (Randerath et al. 1981; Reddy et al. 1984, 1986, 1987). Liver 500 g and 9000 g fractions were analyzed for adducts. There appeared to be several adducts in both the 500 g and 9000 g fractions observed on radioautographs of cellulose-TLC plates. Several adducts were also observed when the 9000 g fraction was studied using HPLC.     

Susceptibility factors and DNA adducts in peripheral blood mononuclear cells of aluminium smelter workers exposed to polycyclic aromatic hydrocarbons

Formation of DNA adducts as a result of exposure to polycyclic aromatic hydrocarbons (PAH) was studied in 98 potroom workers from an aluminium smelting plant and in 55 blue-collar workers without occupational PAH exposure. DNA from peripheral blood mononuclear cells (PBMC) was used for quantitation of individual PAH-DNA adducts by 32P-postlabelling/high performance liquid chromatography (HPLC) analysis. Four individual DNA adducts (denoted A, B, C and D) were quantified in 141 of a total of 153 subjects. Genetic polymorphisms for cytochrome P-4501A1 ( CYP1A1), microsomal epoxide hydrolase, N-acetyltransferase 2, glutathione transferases M1, P1 and T1 ( GSTM1, GSTP1 and GSTT1, respectively) and NAD(P)H: quinone oxidoreductase 1 (NQO1) were analysed. For 52 subjects, analysis of mRNA inducibility of CYP1A1 was performed. No statistically significant differences in the levels of total or individual DNA adducts A, C and D were found between potroom workers and control subjects. All potroom workers and the subgroup of potroom workers who reported to never/sometimes use personal respiratory protection ( n=72) were found to have a significantly higher likelihood of having high levels of adduct B than control subjects [odds ratio (OR) =3.4 with 95% confidence interval (CI) of 1.3-9.2, and OR=4.2 with 95% CI 1.6-11.5, respectively]. In the subgroup, levels of adducts A and B were found to be significantly higher among workers with employment time of less than 6 months ( n=5). Also, the levels of the individual DNA adducts were to some extent modified by genetic polymorphisms in CYP1A1, GSTM1, GSTP1 and NQO1 and by CYP1A1 inducibility. In conclusion, levels of adduct B, identified by 32P-postlabelling/HPLC methodology as an indicator of PAH exposure in aluminium production, were modified by the use of respiratory protection, length of employment and genetic polymorphisms.     

Biomonitoring of diesel exhaust-exposed workers. DNA and hemoglobin adducts and urinary 1-hydroxypyrene as markers of exposure

Diesel exhaust-exposed workers have been shown to have an increased risk of lung cancer. A battery of biomarkers were evaluated for their ability to assess differences in exposure to genotoxic compounds in bus garage workers and mechanics and controls. Lymphocyte DNA adducts were analyzed using the ³²P-postlabelling method with butanol and P1 enrichment procedures. Hydroxyethylvaline (HOEtVal) adducts in hemoglobin were measured by gas chromatography-mass spectrometry (GC-MS) and 1-hydroxypyrene (HPU) in urine determined using HPLC analysis. The exposed workers had significantly higher levels of all three biomarkers compared to the controls. Total DNA adduct levels were 0.84 fmol/μg DNA vs. 0.26 in controls (butanol) and 0.65 fmol/μg DNA vs. 0.08 (P1 nuclease). Median HOEtVal adduct level in exposed workers was 33.3 pmol/g hemoglobin vs. 22.1 in controls. HOEtVal adducts correlated with HPU but not with DNA adducts. The levels of HPU in urine were 0.11 μmol/mol creatinine compared to 0.05 in controls. All three assays applied were sensitive enough to evaluate a low level of exposure to environmental pollutants, with posllabelling and GC-MS as the most sensitive assays. The study indicated that skin absorption of polycyclic aromatic hydrocarbons (PAH) might be an important factor to consider when studying PAH exposure from air pollution sources.     

Relationship between Tissue Concentrations of Polycyclic Aromatic Hydrocarbons and DNA Adducts in Green-Lipped Mussels (Perna viridis)

Green-lipped mussels (Perna viridis) were collected from a site in Hong Kong which is relatively free from polycyclic aromatic hydrocarbon (PAH) contamination, and maintained in situ at this and three other sites with different degrees of PAH contamination. The transplanted mussels were retrieved after a 30-day field exposure. DNA adducts in the gill tissues were quantified, and tissue concentrations of benzo[a]pyrene as well as total PAHs (with potential carcinogenicity) determined for individual mussels. Results indicate that (1) tissue concentration of PAHs and adduct levels in mussels collected from a single site can be highly variable; and (2) adduct levels were related to tissue concentrations of benzo[a]pyrene as well as total PAHs of individual animals.     

Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: studies with the hepatic NADPH:cytochrome P450 reductase null mouse

Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by (32)P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.     

DNA adducts in liver and leukocytes of flounder (Platichthys flesus) experimentally exposed to benzo

In the present study the levels of hydrophobic DNA adducts detected by 32P-postlabelling were followed in liver and leukocytes of flounder (Platichthys flesus) over 10 days following single i.p. injections of two doses of BaP (10 and 50 mg kg(-1) fish weight, respectively). DNA adducts were detected in both tissues of exposed fish 2 days post injection and continued to rise on day 5 and day 10. In flounder exposed to the lower dose of BaP, the levels of hepatic DNA adducts reached higher values on the fifth day compared with flounder exposed to the higher dose. However, at the end of the experiment, the DNA adduct level was again higher in fish from the high dose group compared with the low dose group. There was no substantial increase of DNA adducts in liver of flounder from the low dose group after day 5, while the adduct levels in flounder liver from the high dose group increased throughout the experiment. Earlier studies detecting DNA adducts in BaP-exposed flatfish with the 32P-postlabelling technique have reported declining adduct levels from about 2 days after the exposure, regardless of exposure route. In contrast, the results from our study did not confirm a rapid increase and successive decline of hydrophobic adducts in liver of BaP-exposed flounder.     

Genotoxicity of organic extracts of house dust from Shanxi, China

Indoor combustion of solid fuel such as coal may generate respirable particles containing polycyclic aromatic hydrocarbons (PAH) that may adhere to settled dust. Dust might therefore present a major source of PAH exposure in humans. This study evaluated the in vitro and in vivo genotoxicity of PAH mixtures extracted from house dust samples. Four dust samples (E1-4) were collected from houses in Shanxi, China, where coal is heavily used for heating and cooking. For comparison, a coal sample was also collected from one of the houses and included in the analyses. The samples were extracted with methylene chloride:acetone (95:5 v/v), dried, and redissolved in appropriate solvents for assessment in genotoxicity assays. Samples were evaluated for their ability to induce point mutations in bacteria and DNA adducts in vivo. DNA adduct levels were analyzed by nuclease P1-enhanced 32P-postlabeling. PAH were quantified using gas chromatography/mass spectrometry. Based on chemical analysis, sample E1 had the highest concentration by sampling area of benzo[a]pyrene (BaP) (181 microg/m2) and total PAH (10100 microg/m2). However, based on the microbial genotoxicity assay, sample E3, with the highest carcinogenic PAH/total PAH ratio (26%), produced the greatest number of revertants. In mice, administration of the extract of coal induced more adducts (9.81 adducts per 10(9) nucleotides) than dust extracts. The results of this study confirm the presence of genotoxic chemicals in residential dust. Inhalation of respirable particles containing similar mixtures of PAH represents a cancer risk for humans.     

Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes

Ellipticine is a potent antineoplastic agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). We examined a panel of genetically engineered V79 cell lines including the parental line V79MZ and recombinant cells expressing the human CYP enzymes CYP1A1, CYP1A2 or CYP3A4 for their ability to activate ellipticine. The extent of activation was determined by analysing DNA adducts by 32P-postlabelling. Ellipticine was found to be toxic to all V79 cell lines with IC(50) values ranging from 0.25 to 0.40 microM. The nuclease P1 version of the 32P-postlabelling assay yielded a similar pattern of ellipticine-DNA adducts with two major adducts in all cells, the formation of only one of which was dependent on CYP activity. This pattern is identical to that detected in DNA reacted with ellipticine and the reconstituted CYP enzyme system in vitro as confirmed by HPLC of the isolated adducts. Total adduct levels ranged from 2 to 337 adducts per 10(8) nucleotides, in the parental line and in V79 expressing CYP3A4, respectively. As in vitro, human CYP1A2 and CYP1A1 were less active. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in cells in culture, and confirm the formation of covalent DNA adducts as a new mechanism of ellipticine action.     

Development of a 32P-postlabelling method for the detection of 1,N 2-propanodeoxyguanosine adducts of crotonaldehyde in vivo

Crotonaldehyde is a genotoxic, mutagenic and carcinogenic alpha,beta-unsaturated carbonyl compound which forms 1,N2-propanodeoxyguanosine adducts. Humans are exposed to this compound at work places, and from tobacco smoke and air pollution, but also from food and beverages. Therefore crotonaldehyde can play a significant role in carcinogenesis. Since in vivo measurement of DNA adducts of crotonaldehyde can improve cancer risk assessment and contribute to the clarification of the role of crotonaldehyde in carcinogenicity, we developed, adapted and optimized a 32P-postlabelling technique for the adducts of crotonaldehyde based on nuclease P1 enrichment and on a polyethylene imine modified cellulose TLC to provide a detection sensitivity of three adducts per 10(9) nucleotides and a labelling efficiency of 80-90%. We also report a readily performable synthesis of adduct standards and demonstrated that DNA is completely digested to the 3'-monophosphate nucleotides under the conditions of our enzymatic DNA hydrolysis. We showed that the postlabelling method developed is appropriate for in vivo DNA-binding studies. Female Fischer 344 rats were treated by gavage with crotonaldehyde at doses of 200 and 300 mg/kg body weight, and 20 h after treatment adduct levels of 2.9 and 3.4 adducts per 10(8) nucleotides, respectively, were found in the liver DNA. Only 1.6 nucleotides per 10(8) nucleotides were found 12 h after treatment at 200 mg/kg body weight. Absolutely no adducts could be found in liver DNA of untreated rats with our method at the detection limit of three adducts per 10(9) nucleotides. In contrast to our group, the group of Chung have reported crotonaldehyde adduct levels in the range of 2.2 22 adducts per 10(8) nucleotides in DNA of untreated Fischer 344 rats. The clarification of this discrepancy is of importance for the elucidation of the role of crotonaldehyde in carcinogenicity, and both groups have decided to clarify this in cooperation in the near future.     

PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure: a laboratory experiment with Atlantic cod

In order to perform environmental risk assessments with regard to oil contamination in the sea, it is important to obtain knowledge about threshold levels for possible adverse effects in marine organisms. With this objective in mind, selected biomarkers were studied in Atlantic cod (Gadus morhua) chronically exposed to mechanically dispersed crude oil. The fish were exposed for 30 days in a continuous flow system to nominal concentrations of 0.06, 0.25 and 1 ppm North Sea crude oil. Fish were sampled five times during the exposure period. In addition, the 1 ppm group and the control group were sampled 1 week after the end of exposure. Polyaromatic hydrocarbon (PAH) concentrations in the seawater were analysed regularly by direct fluorescence and, at one occasion, by gas chromatography with mass spectrographic detection (GC/MS) measurements. Liver samples were analysed for parent PAH levels by means of GC/MS measurements, and PAH metabolites in bile were analysed by means of fixed wavelength fluorescence. Cytochrome P450 induction in liver was estimated by ethoxyresorufin-O-deethylase (EROD) activity, and hepatic DNA adducts were analysed by the 32P-postlabelling assay. The parent PAH concentrations in liver showed peak levels 3 days after the start of exposure, followed by a reduction towards the end of the experiment. In contrast, the PAH metabolites in bile and EROD activity showed generally increasing levels throughout the whole exposure period, indicating an increased biotransformation efficiency. The level of DNA adducts in the 1 ppm group showed a stable increase during the entire exposure period. Only a slight, non-significant decrease in DNA adduct levels was observed after 7 days of recovery in clean water. Exposure-dependent responses were observed for all three biomarkers. The lowest nominal concentration of dispersed oil in water, 0.06 ppm, corresponded to a measured total PAH concentration in the water of 0.3 ppb. Atlantic cod exposed to this concentration showed increased levels of PAH metabolites in bile and a slight induction of CYP1A, as well as formation of DNA adducts when compared with control fish. Particularly noteworthy is the detection of DNA adducts at such a low exposure concentration of oil in water, which, to our knowledge, is a novel finding. These dose-response data may serve as useful contributions when assessing environmental risk with regard to marine oil pollution.     

Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: field and laboratory investigations

Hg bioaccumulation was investigated in the king scallop Pecten maximus in the laboratory and in the field. In controlled conditions, scallops were exposed to (203)Hg through seawater, sediment and food in order to determine its uptake and depuration kinetics. In the field, Hg and metallothionein (MT) concentrations and the metal subcellular distribution were determined in scallops from two sites of the Bay of Seine (France) differently subjected to the Seine river inputs. While Hg concentrations in the whole soft parts and kidneys (viz. the highest accumulator organ) did not differ between scallops from both sites (74-156 ng g(-1)dry wt), they did for the digestive gland and the gills. According to the experimental results, a higher exposure to dissolved Hg might occur in the site close to the estuary whereas Hg would be mainly incorporated via the dietary pathway in the site away from the estuary. Within the cells of wild scallops, Hg was mainly associated to the cytosolic fraction in the digestive gland and gills (60-100%). However, the lack of relationship between Hg and MT levels suggests that Hg detoxification in P. maximus involves other, non-MT, soluble compounds. In kidneys, insoluble compounds played an important role in Hg sequestration. No effect of scallop age was observed neither on Hg and MT concentrations nor on the subcellular distribution of the metal. Finally, according to FAO/WHO recommendations (maximum weekly Hg intake), our results clearly indicate that the low Hg contents in the edible part of the king scallops from the Bay of Seine prevent any risk for human consumers.     

The environmental pollutant and carcinogen 3-nitrobenzanthrone induces cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in rat lung and kidney, thereby enhancing its own genotoxicity

3-Nitrobenzanthrone (3-NBA) is a carcinogen occurring in diesel exhaust and air pollution. Using the (32)P-postlabelling method, we found that 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), are activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney. Each compound generated identical five DNA adducts. We have demonstrated the importance of pulmonary and renal NAD(P)H:quinone oxidoreductase (NQO1) to reduce 3-NBA to species that are further activated by N,O-acetyltransferases and sulfotransferases. Cytochrome P450 (CYP) 1A1 is the essential enzyme for oxidative activation of 3-ABA in microsomes of both organs, while cyclooxygenase plays a minor role. 3-NBA was also investigated for its ability to induce NQO1 and CYP1A1 in lungs and kidneys, and for the influence of such induction on DNA adduct formation by 3-NBA and 3-ABA. When cytosols from rats treated i.p. with 40mg/kg bw of 3-NBA were incubated with 3-NBA, DNA adduct formation was up to 2.1-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. Incubations of 3-ABA with microsomes of 3-NBA-treated rats led to up to a fivefold increase in DNA adduct formation relative to controls. The stimulation of DNA adduct formation correlated with the potential of 3-NBA to induce protein expression and activity of CYP1A1. These results demonstrate that 3-NBA is capable to induce NQO1 and CYP1A1 in lungs and kidney of rats thereby enhancing its own genotoxic and carcinogenic potential.     

Urinary excretion and DNA binding of coal tar components in B6C3F1 mice following ingestion

Urinary excretion of polycyclic aromatic hydrocarbon (PAH) metabolites and DNA binding of coal tar components in male mice were investigated following the ingestion of a coal tar adulterated diet. Male B6C3F1 mice were able to tolerate an F0927 basal gel diet which contained from 0.1 to 1% coal tar (tar weight/dry food weight) for 15 days. Mice maintained on a 0.1 and 0.2% coal tar diet had body weight gains similar to those of control animals. However, mice maintained on the 0.5 and 1.0% diet had body weight gains considerably lower than control values. Chemical-DNA adduct formation was detected and quantified in lung and forestomach tissue of animals on 0.1, 0.2, 0.5, and 1% coal tar containing diets. A dose-related effect was observed in lung DNA adduct formation while no dose effect was observed in forestomach tissue. In addition, overall adduct levels in lung tissue were considerably higher than forestomach levels for animals on the 0.5 or 1% diet. In contrast, DNA adduct levels were highest in the forestomach of animals on diets lower in coal tar content (0.1 or 0.2%). Chemical-DNA adducts of coal tar components were also evaluated for four other coal tar samples which varied in chemical composition. Mice were maintained on diets containing 0.25% of each coal tar for 15 days. Chemical-DNA adducts were detected in lung, liver, and spleen for all animals receiving these coal tar diets. DNA adduct patterns were similar while quantitative differences were observed between coal tar samples and tissue sites. Highest adduct levels were detected in lung DNA. Benzo[a]pyrene content in coal tar samples could not account for the DNA adduct levels observed with coal tar ingestion. The urinary excretion of select PAH metabolites following coal tar ingestion was evaluated by using urine collected on days 1 and 14 of diet administration. The levels of 1-hydroxypyrene in urine, the major PAH metabolite detected, correlated with the pyrene content of these coal tars. These data demonstrate that coal tar components are readily bioavailable following ingestion.     

Genotoxic activity and induction of biotransformation enzymes in two human cell lines after treatment by Erika fuel extract

On 12 December 1999, the tanker Erika broke in two parts at about 60km from the Brittany French coasts (Point of Penmarc'h, Sud Finistère, France). About 10,000tonnes of heavy oil fuel were released in the sea. DNA adduct have been detected in fish liver and mussels digestive gland exposed to the Erika oil spill. In order to investigate the mechanism by which Erika fuel extract exhibits genotoxic effects the induction of DNA adducts by an Erika fuel extract have been analysed on two cell lines, human epithelial bronchial cells (WI) and human hepatoma cells. DNA adducts, reflected by a diagonal radioactive zone and individual adducts are detected only in hepatoma cells indicating biotransformation via CYP 1A2 and CYP 1B1. In addition, Erika fuel extract induces some metabolizing enzymes such CYP 1A2, COX2 and 5-LOX, the two later are involved in cancer processes. Formation of leucotrienes B4 (LTB(4)), a mediator playing a role in inflammation, is induced in epithelial bronchial cells. Since inhalation is one of the ways of contamination for human, the above results are important for human health and prevention.     

Effect of a standardized complex mixture derived from coal tar on the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons in human cells in culture

A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, standard reference material (SRM) 1597, has been shown to initiate tumor formation in mouse initiation-promotion assays in our laboratory [(2001) Carcinogenesis 22 (7), 1077-1086]. To determine the effects of SRM 1597 on PAH activation in human cells, we investigated the PAH-DNA adduct formation in the human mammary carcinoma-derived cell line MCF-7. We examined the effects of SRM 1597 on the metabolic activation to DNA binding derivatives of two carcinogenic PAHs, the bay region containing benzo[a]pyrene (B[a]P) and the more carcinogenic fjord region containing dibenzo[a,l]pyrene (DB[a,l]P). PAH-DNA adduct analysis by 33P-postlabeling and reversed phase high-performance liquid chromatography revealed a significant decrease in the levels of both B[a]P and DB[a,l]P DNA adduct formation on cotreatment with SRM 1597 in comparison to cells exposed to B[a]P or DB[a,l]P alone. However, the inhibition of PAH-DNA adduct formation only occurred within the first 48 h of exposure in cells cotreated with SRM 1597 and B[a]P. In contrast, SRM 1597 significantly inhibited the level of DB[a,l]P DNA adducts throughout the 120 h of exposure. Induction of human cytochrome P450 (P450) enzymes 1A1 and P4501B1 on treatment with SRM 1597 was observed by immunoblots. These results suggest that the important factors in determining the carcinogenic activity of PAH within a complex mixture would depend on the ability of other components of the mixture to promote or inhibit the activation of carcinogenic PAH by the induction of P450 enzymes followed by the formation of DNA adducts.     

Genotoxicity related to transfer of oil spill pollutants from mussels to mammals via food

Heavy fuel oils containing high levels of polycyclic aromatic hydrocarbons (PAHs) were released into the marine environment after the Erika oil spill on the Atlantic coast. As highly condensed PAH pollutants can bioaccumulate in invertebrates, their transfer to vertebrates through the food chain was of concern. This study aimed to estimate potential genotoxic effects in rats fed for 2 or 4 weeks with the marine mussel Mytilus edulis contaminated by oil pollutants. Two levels of PAH contamination were studied, around 100 and 500 microg of total PAHs/kg dry weight (d.w.) in mussels. Genotoxic damage in rats was investigated by single-cell gel electrophoresis (Comet assay) and micronucleus assays in liver, bone marrow, and peripheral blood. DNA damage was observed in the liver of rats fed with the most contaminated mussels (500 microg PAHs/kg d.w.).DNA damage also was observed in the bone marrow but less than that in the liver. A small increase in micronuclei frequency was registered as well. This work underlines the bioavailability of pollutants in fuel-oil-contaminated mussels to consumers and the usefulness of the Comet assay as a sensitive tool in biomonitoring to analyze responses to PAH transfer in food. The occurrence of substituted PAHs and related compounds such as benzothiophenes in addition to nonsubstituted PAHs in fuel oils and mussels raised the question of whether they were implicated in the genotoxic effects registered in rats.     

Levels of PAH–DNA adducts in placental tissue and the risk of fetal neural tube defects in a Chinese population

We examined the relationship between PAH–DNA adduct levels in the placental tissue, measured by a highly sensitive ³²P-postlabeling assay, and the risk of fetal neural tube defects (NTDs). We further explored the interaction between PAH–DNA adducts and placental PAHs with respect to NTD risk. Placental tissues from 80 NTD-affected pregnancies and 50 uncomplicated normal pregnancies were included in this case-control study. Levels of PAH–DNA adducts were lower in the NTD group (8.12 per 10⁸ nucleotides) compared to controls (9.92 per 10⁸ nucleotides). PAH–DNA adduct concentrations below the median was associated with a 3-fold increased NTD risk. Women with a low PAH–DNA adduct level in concert with a high placental PAH level resulted in a 10-fold elevated risk of having an NTD-complicated pregnancy. A low level of placental PAH–DNA adducts was associated with an increased risk of NTDs; this risk increased dramatically when a low adduct level was coupled with a high placental PAH concentration.